NEW METHOD.

专利摘要:
This invention relates to a method of producing a virus in cell culture comprising at least the steps of a) using a population of cells cultured in a cell culture medium, b) infecting the population of cells with i. inoculation of the population with the virus, and ii. incubating the inoculated population so as to allow the virus to replicate and spread, c) collect the virus produced, thereby obtaining a viral harvest, and d) purify the virus. An input power density of at least 15 W / m3, at least 30 W / m3, at least 60 W / m3, at least 100 W / m3, at least 120 W / m3 being applied to the cell culture at least during step b).
公开号:BE1022132B1
申请号:E2014/0635
申请日:2014-08-28
公开日:2016-02-19
发明作者:Pascal Charles Louis Gerkens；Michèle Thérèse Rita Lecocq；Tatsuya Kasugaya；Kenjiro Kawatsa；Yoshinobu Miyatsu；Tetsuro Tanabe
申请人:Glaxosmithkline Biologicals S.A.；The Chemo-Sero-Therapeutic Research Institute；
IPC主号:

专利说明:

NEW METHOD
DECLARATION CONCERNING RESEARCH SPONSORED BY THE GOVERNMENT.
This invention was developed with the support of the US Government under contract No. HHS0100200600011C to the Department of Health and Social Services; the US government having certain rights to the invention.
TECHNICAL AREA
The present invention relates to a method for producing virus, or viral antigens, in cell culture, viruses or viral antigens obtainable by this method and vaccines containing said viruses or viral antigens. In particular, the invention relates to a method for improving the viral yield. BACKGROUND
The development of cell culture techniques as an alternative to conventional egg-based production systems for the production of viral vaccines is probably the fastest and most promising solution to overcome the disadvantages and constraints associated with conventional systems based on the use of eggs. Commercial production of viral vaccines typically requires large amounts of virus as a source of antigen. However, the egg-based process is vulnerable because of the variable biological quality of the eggs and its lack of logistical flexibility as large quantities of suitable eggs are not available.
Systems in cell culture appear to be an appropriate alternative method of vaccine preparation, which is simpler, more flexible, and more regular, thus improving the possibilities of evolution of vaccine production capacities and consequently of obtaining large quantities of vaccines. virus, if necessary, for example, in response to a natural pandemic threat or terrorist attack.
Effective vaccine production, however, requires large-scale virus culture at high yields from a host system. The culture conditions in which a virus is cultured have an important significance in achieving an acceptable high yield of the virus. Therefore, to increase the yield of the desired virus, both the system and the culture conditions must be specifically adapted to create an advantageous environment suitable for large scale production of the desired virus. One way of doing this is to improve the specific productivity of the cells, for example, by improving the culture medium, or by increasing the cell density. Knowing that after production, the virus produced in cell culture must be recovered from the cell culture and purified, another way to improve viral yield is to limit the loss of viral material occurring during the different stages of the virus. purification. Therefore, there remains a need for alternative and improved methods for producing viruses at increased viral yield. The method according to the present invention makes it possible to obtain a better viral yield compared to the methods known in the state of the art.
SUMMARY OF THE INVENTION
According to a first aspect, the invention relates to a method for producing a virus in cell culture comprising at least the steps of: a) using a population of cells in a cell culture medium,. b) infect the cell population by: i. inoculation of the population with the virus, and ii. incubation of the inoculated population to allow the virus to replicate and spread, c) collect the virus produced, thereby obtaining a viral harvest, and d) purify the virus, in which an input power density of at least 15 W / m3, at least 30 W / m3, at least 60 W / m3, at least 100 W / m3, or at least 120 W / m3 is applied to the cell culture at least least during step b).
In a second aspect, the invention relates to a process for preparing a vaccine comprising at least the step of mixing the virus obtained according to the method of the present invention with a pharmaceutically acceptable carrier. , " DETAILED DESCRIPTION '
This invention relates to an improved method of producing virus in cell culture, which is particularly useful for large scale production. In particular, the method according to the invention contributes to increasing the viral yield by limiting the viral loss during the purification process. Surprisingly, the inventors have observed that certain changes occurring in the upstream part of the virus production process, such as the increase of the input power density applied to the cells cultured to produce a virus, resulted in a significant improvement in the downstream part of said process, such as an increase in the viral yield obtained after several purification steps. In particular, the inventors. observed that, while the increase in input power during the upstream cell culture phase had no impact on the specific productivity of the cells, a positive effect was obtained in the subsequent step of clarification by microfiltration in that the viral yield obtained after said step was significantly increased, compared to the yield obtained when a lower input power was applied during the cell culture phase. In addition, not only did the recovery of the virus after the microfiltration step increase, but also the percentages of recovery obtained were more regular and less variable from one experiment to another. The inventors have also observed a similar beneficial effect of the increase in the input power density during the upstream cell culture phase on a subsequent sucrose gradient ultracentrifugation step implemented during the downstream purification phase of the invention. virus. In particular, the inventors have observed that the increase in input power density applied to the cultured cells makes it possible to increase, by a factor of approximately 2, the load capacity of the rotor used for the ultracentrifugation step. on sucrose gradient. Therefore, the virus yield obtained after several purification steps led to a better overall yield of the virus at the end of the process of producing a virus. Unexpectedly, the inventors have observed that a higher input power density does not cause harm or damage to the cells.
The method according to the present invention offers the advantage of being easy to implement, requiring neither additional step nor equipment other than that typically used as standard to produce a virus in culture. cellular.
"Carrying capacity" is the quantity in liters of viral harvest collected during step c) of the process according to the invention which is charged per liter of rotor of the centrifuge used for the sucrose gradient ultracentrifugation step, or the "equivalent" amount in liters of virus harvest when said viral harvest has been treated before being subjected to the sucrose gradient ultracentrifugation step, as part of previous purification steps. For example, as described below, after being collected, the virus flake may be subjected to ultrafiltration / diafiltration before being subjected to a sucrose gradient ultracentrifugation step, said ultrafiltration / diafiltration typically resulting in a concentration of the viral harvest, ie, the quantity in liters of viral harvest, after this step.
"Input power density" is the amount of power per unit volume, or the average speed of specific energy dissipation. In cell culture, this corresponds to the amount of power communicated to a volume. of cell culture through the shaft and turbines of the agitator. It is expressed in W / m3. The empirical formula used in the state of the art to calculate the input power values is: P / V = (Np x n3 X di5) / V, where Np is the turbulent power number of the turbine, n is the stirring speed measured in revolutions per second of the turbine, di is the diameter of the turbine measured in meters and V is the volume of culture in cubic meters.
The method according to the invention is applicable to all types of cells, whether they are adherent cells grown on microcarriers or cells in suspension. Therefore, in the context of the present invention, the term "cell suspension" may designate both adherent cells grown on microcarriers and cells capable of developing in suspension, ie which do not require adherent support, such as micro-media, to develop.
Typically, the input power density is applied by mechanical movement of the cell suspension. Said mechanical movement can be obtained by various means. For example, the mechanical movement of the cell suspension can be achieved by means of a stirring device, such as turbines. The agitation can be imparted as an axial flow, a radial flow, or a combination of both, depending on the type of turbine used. Typically, the turbines can be divided into several groups according to their geometry, and in particular, the geometry of their blades: (i) flat-blade turbines, also called Rushton turbines or Rushton-type turbines; (ii) inclined blade turbines; and (iii) submarine blade turbines.
Flat-blade turbines, or Rushton turbines, consist of flat blades that are mounted vertically along the agitator shaft, thereby producing a unidirectional radial flow. These types of turbines are commonly used in fermentations of cell lines that are not considered sensitive to shear, such as yeasts, bacteria, and certain fungi. However, as described herein, the inventors have found that Rushton turbines can also be suitably used with animal cells according to the method of the present invention.
The inclined blade turbines consist of flat blades which are mounted at an angle to the agitating shaft, thereby producing a simultaneous axial and radial flow. These inclined blade turbines are often used with shear-sensitive cell lines that develop in suspension or with microcarriers. These turbines are often used in batch or semi-batch cultures, but they can also be used in continuous and infusion mode.
The front face of the blades on a submarine blade turbine may be flat, or concave, while their rear face is convex, thus producing a unidirectional axial flow.
Different factors need to be considered in selecting the appropriate types of turbines, such as cell type, type of cropping system (batch, semi-batch or infusion), type of culture reactor, and level of culture. desired input power density. It is within the skill of those skilled in the art to determine and select the appropriate turbine to use and this according to the specific conditions above. In one embodiment, an input power density, in particular an input volumetric power ranging from 30 to 120 W / m.sup.3, applied in the method according to the invention is obtained by means of a turbine with blades. inclined. In another embodiment, an input power density, in particular an input power density greater than 120 W / m.sup.3, applied in the method according to the invention is obtained using a Rushton turbine.
The input power density applied to animal cell cultures is typically much lower than for microbial cultures because of the supposedly higher fragility of animal cells that lack protective cell walls, which makes them particularly sensitive shear stress and foam formation as microbial cultures. However, as observed by the present inventors, the application of a high input power density, to a certain extent, to cultures, of animal cells used to produce a virus is not detrimental to the cells, while by favorably promoting the viral yield during the purification of the virus produced.
The method according to the invention can be implemented in a wide range of input power density values.
"Maximum value" means the maximum input power density that can be applied in the absence of an overall negative impact on viral yield. A factor known to have a possible negative impact on viral yield is cell integrity, such as reduced cell viability. The skilled person knows how to monitor cell viability. A non-limiting example of suitable methods is cell staining (which discriminates living cells from dead cells), such as trypan blue staining, or any other appropriate staining known. Alternatively, cell viability can also be evaluated and measured by flow cytometry, such as, for example, by FACS (fluorescence activated cell sorting). In addition, when defining the maximum value, the specific productivity of the virus cells can be taken into account. By "cell specific productivity" is meant the ability of the cells to produce the virus, i.e. the amount of virus obtained at the collection step c), before being subjected to purification. Preferably, the input power density applied to the cultured cells will not be able to affect the specific productivity of the virus cells.
"Minimum value" of the input power density means the minimum value providing an improvement in the viral yield during the virus purification phase, compared to the viral yield obtained during the purification phase of the virus when none, or a low input power density is applied to the cultured cells. Any method known in the art for measuring virus yield or viral titer can be suitably used to help determine the optimum input power density values to be applied in the method of the invention. For example, CPE (cytopathic effect) can be measured by monitoring morphological changes in host cells after virus inoculation, including rounding, disorientation, swelling or shrinkage, death, and detachment of the surface of the cells. In addition, the detection of a specific viral antigen can be monitored by standard protein detection techniques, such as Western-Blot analysis, at any time after the cells are inoculated with the virus of interest must be produced. In the particular case of influenza virus, the HA content can be monitored at any time after inoculation of the cells with the virus, by SRD assay (Wood, JM, et al (1977) J. Biol Standard. 5, 237-247), which is a technique known to those skilled in the art. In addition, the SRD assay can also be used at any time during the purification phase to evaluate the viral yield before and after any given purification step. According to the method of the present invention, the input power density applied to the animal cells to produce the viruses is typically 15 to 900 W / m 3, advantageously 30 to 500 W / m 3, more preferably 60 to 250 W / m3, or even 120 to 200 W / m3. In one embodiment, the input power density value used in the process according to the invention is at least 15 W / m 3, at least 30 W / m 3, at least 60 W / m 3, at least 100 W / m3, or at least 120 W / m3. In a specific embodiment, the input power density value used in the method according to the invention is 30 W / m3. Alternatively, in another specific embodiment, the input power density value used in the method according to the invention is 120 W / m3.
The process according to the invention is applicable to any type of culture reactor, of any size, such as, for example, flasks, bottles for rotary stirrer, or bioreactors, as long as said reactors are able to receive cooling devices. stirring, such as for example turbines, or are compatible with the use of a separate stirring device. It is not necessary that the reactor is equipped with a stirring device. The stirring device can be separated from the reactor itself. For example, in the case of single-use bioreactors, typically consisting of plastic bags, such as Wave ™ bioreactors, agitation of said single-use bioreactors can be achieved by placing the bioreactors on a stirring table, either an agitator shaker with orbital movement or an agitator shaker with axial movement. The bioreactors may be based on any type of material, such as glass or stainless steel for non-single use bioreactors, or plastic for single use bioreactors. In addition, the bioreactors can be of any shape, such as cylindrical or cubic. Bioreactors are typically used for intermediate scale production, such as 1-10 L, and large scale production, such as 20-1000 L, and beyond.
The process according to the invention is applicable to any type of bioreactor of any size. In particular, the process according to the invention is suitable for 10 L, 200 L, 500 L, 1000 L or 10,000 L reactors. A particularly suitable type of bioreactor that can be used in the process according to the invention is that of bioreactors. stirred tank operating in batch or continuous mode. Single-use bioreactors are a type of suitable alternative bioreactors that can be used in the process of the invention. In one embodiment, the cells used in the method according to the invention are cultured in a disposable bioreactor of 200 L.
According to the invention, the cells can be cultured in various ways, such as, for example, using discontinuous, semi-batch, or continuous systems, such as perfusion systems. Infusion is particularly advantageous when a high cell density is desired. A high cell density may be particularly advantageous for maximizing the amount of virus that can be produced from a given cell type. The method of producing a cell culture virus in which a high input power density is applied to the cell culture according to the present invention is applicable to any of the above systems, regardless of whether the cells are cultured at least once. discontinuous, semi-batch or continuous mode. In one embodiment according to the invention, the cells used in the process according to the present invention are cultured in batch mode. '- ·
The method according to the invention can be applied to a wide range of viruses, namely any virus capable of infecting cells and to use them for its replication, comprising, inter alia, adenoviruses, hepadnaviruses, herpesviruses, orthomyxoviruses. , papovaviruses, paramyxoviruses, picornaviruses, poxviruses, reoviruses and retroviruses. In particular, the method according to the invention is applicable to enveloped viruses, such as myxoviruses. In one embodiment, the viruses produced by the process according to the invention belong to the family of orthomyxoviruses, in particular the influenza virus (influenza).
Viral viruses or antigens can be derived from an orthomyxovirus, such as the influenza virus. The orthomyxoviral antigens may be selected from one or more of the viral proteins, including haemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein (M1), membrane protein (M2), or more of the transcriptases (PB1, PB2 and PA). In particular, particularly suitable antigens include HA and NA, the two surface glycoproteins that determine the antigenic specificity of influenza subtypes.
The influenza virus may be selected from the group of human influenza virus, avian influenza virus, equine influenza virus, swine flu virus, and feline influenza virus. The influenza virus is more particularly chosen from strains A, B and C, preferably from strains A and B.
Influenza antigens can be derived from interpandemic influenza strains (annual or seasonal). Alternatively, influenza antigens can be derived from strains that have the ability to cause a pandemic rash (i.e., influenza strains with
rO new haemagglutinin compared to haemagglutinin in commonly circulating strains, or influenza strains that are pathogenic in avian subjects and have the power to be transmitted horizontally in the human population, or influenza strains that are pathogenic to humans). Depending on the season and the particular nature of the antigen included in the vaccine, the antigens of the flu can be derived from one or more of the following hemagglutinin subtypes: H1, H2, H3, H4, H5 , H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16. Preferably, the influenza virus or its antigens come from subtypes H1, H2, H3, H5, H7 or H9.
In one embodiment, the influenza viruses come from the subtypes H2, H5, H6, H7 or H9. In another embodiment, the influenza viruses are from H1, H3 or B.
The cells that are used in the method according to the invention can in principle be any desired type of animal cells that are cultured in cell culture and capable of supporting the replication of the virus. It can be either primary cells or continuous cell lines. Genetically stable cell lines are preferred. Mammalian cells are particularly suitable, for example, human, hamster, cattle, monkey or dog cells. Alternatively, insect cells are also suitable, such as, for example, SF9 cells or Hi-5 cells.
A number of mammalian cell lines are known in the art and include PER.C6 cells, HEK cells, human embryonic kidney cells (293 cells), HeLa cells, CHO cells, Vero cells and MDCK cells.
Suitable monkey cells are, for example, African green monkey cells, such as kidney cells, as in the Ver cell line. Suitable dog cells are, for example, kidney cells such as the MDCK cell line. .
Mammalian cell lines suitable for cultivating the influenza virus include MDCK cells, Vero cells, or PER.C6 cells. These cell lines are all widely available, for example, from the ATCC (American Type Cell Culture) collection.
According to a specific embodiment, the method according to the invention uses MDCK cells. The original MDCK cell line is available from ATCC under the name CCL-34, but derivatives of this cell line can also be used, such as MDCK cells adapted for suspension culture (WO 1997/37000).
Alternatively, the cell lines useful in the invention can be derived from avian sources, such as chicken, duck, goose, quail or pheasant. Avian cell lines can be derived from various stages of development including embryos, chicks and adults. In particular, the cell lines can be derived from embryonic cells, such as embryonic fibroblasts, germ cells, or individual organs, including neuronal, brain, retinal, renal, hepatic, cardiac, muscle tissues and membranes, or extraembryonic protecting the embryo. Chicken embryo fibroblasts (CEF) can be used. Examples of avian cell lines include avian embryonic stem cells (WO01 / 85938) and retinal duck cells (WO05 / 042728). In particular, the EB66® cell line derived from duck embryonic stem cells is contemplated in the present invention (WO08 / 129058). Other suitable avian embryonic stem cells include the EBx® cell line derived from chicken embryonic stem cells EB45, EB14 and EB14-074 (WO2006 / 108846). This EBx® cell line has the advantage of being a stable cell line, obtained naturally without the need for genetic, chemical or viral modification. These avian cells are particularly suitable for the culture of influenza viruses.
According to a particular embodiment, the method according to the invention uses EB66® cells.
The cell culture conditions (temperature, cell density, pH value, etc.) vary over a very wide range due to the adaptability of the cells used and can be adapted to the requirements of the particular crop specific conditions of the viruses. It is within the skill of those skilled in the art to determine the appropriate culture conditions, since cell culture is very widely documented in the state of the art (see, for example, Tissue Culture, Academic Press, Kruse and Paterson, Editors (1973), and RI Freshney, Culture of animal cells: A manual of technical basic, Fourth Edition (Wiley-Liss Inc., 2000, ISBN 0-471-34889-9).
In a specific embodiment, the cells used in the method described in the present invention are grown in serum-free and / or protein-free media. A "serum-free medium" (SFM) refers to a ready-to-use cell culture medium that does not require the addition of serum for cell survival and growth. This medium is not necessarily chemically defined and may contain hydrolysates of various origins, plant for example. The serum-free medium has the advantage that contamination by chance viruses, mycoplasmas or other unknown infectious agents can be avoided. "Protein Free" refers to cultures in which cell multiplication occurs in the absence of proteins, growth factors, other protein additives and non-serum proteins. Eventually trypsin or other proteases that may be necessary for virus growth may be added during viral infection. The cells growing in this culture naturally contain proteins themselves.
Serum-free media are commercially available from many sources, for example, SFM VP (Invitrogen Ref 11681020), Opti-Pro (Invitrogen, Ref 12309-019), or EX-CELL (JHR Bioscience). . .
Prior to virus infusion, the cells are cultured at about 37 ° C, more preferably at 36.5 ° C, at a pH of 6.7 to 7.8, advantageously around 6.8-7. , 5, and better still around 7.2. When the cells are insect cells, the temperature before virus infection typically ranges from 25 to 29 ° C, and is appropriate at 27 ° C.
According to the method of producing a virus according to the invention, the production of virus in cell culture generally comprises the steps of a) using a population of cells cultured in a culture medium, b) inoculating said cells with the virus of interest to produce, so as to initiate the process of infection of the cells by the virus, and incubate or cultivate the inoculated cells for a desired period of time to allow replication and spread of the virus, and c) collect the virus product. The high input power density according to the present invention is advantageously applied. during at least step b), and more preferably, during steps a) and b). Therefore, according to one embodiment, in the method of producing a virus according to the present invention, an input power density of at least 15 W / m 3, of at least 30 W / m 3, of at least 60 W / m 3, at least 100 W / m 3, or at least 120 W / m 3 is applied to the cells cultured during steps a) and b).
The terms "inoculate / inoculate" and "inoculated cells" are meant within the scope of the present invention. as the time when the virus of interest is added to the cells, and denote the cells to which the virus of interest was added, respectively. The terms "after inoculation" are understood as the time after the virus has been added to the cells. In the remainder of the description, the time "after inoculation" is indicated in minutes, hours or days, such as "2 h after inoculation", or "1 day (J1) after inoculation". The day the cells are inoculated with the virus is considered as day 0 (OJ).
The three successive stages of virus inoculation, virus replication, and virus spread are all part of the broader process of virus infection. High input power density as defined in the present invention may be applied to the cell culture during any of the above steps. The high input power density in the sense of the present invention can be suitably applied. cells as they develop and are cultured before being infected with the virus of interest. The high input power density in the sense of the present invention may also be suitably applied to cells after they have been inoculated with the virus and / or during their incubation to allow the virus to replicate and propagate. The high input volumetric power can also be appropriately applied to the cells as they grow and are cultured before being infected, after they have been inoculated with the virus, and during their incubation to allow the virus will replicate and spread. In one embodiment, the input power density is applied to the cells used in the method according to the invention after they have been inoculated with the virus of interest and until the virus produced is collected. In another embodiment, the high input power density is applied to the cells as they grow and are cultured before being infected with the virus of interest, as well as after they have been infected. inoculated with said virus and until the virus produced is collected. In order to produce large amounts of virus, it may be advantageous to inoculate the cells with the virus of interest once the cells have reached a high density. Generally, inoculation is practiced when the cell density is at least about 5 x 10 6 cells / ml, advantageously 6 x 10 6 cells / ml, more preferably 7 x 10 6 cells / ml, more preferably 8 x 106 cells / ml, more preferably 9 x 10 6 cells / ml or more, such as 10 x 10 6 cells / ml, 11 x 10 6 cells / ml, 12 x 10 6 cells / ml or 13 x 10 6 cells / ml or more, as 20 x 10 6 cells / ml, 25 x 10 6 cells / ml, or 30 x 10 6 cells / ml. In one embodiment of the present invention, the cell density achieved before virus infection occurs is at least 8 x 10 6 cells / ml, 9 x 10 6 cells / ml, 10 x 10 6 cells. / ml, 11 x 10 6 cells / ml, 12 x 10 6 cells / ml or 13 x 10 6 cells / ml. In another embodiment, the cell density achieved before virus infection occurs is at least 20 x 10 6 cells / ml, 25 x 10 6 cells / ml, or 30 x 10 6 cells / ml. These high density levels can. to be advantageously achieved by means of a perfusion system for cell culture. The optimal cell density to achieve the highest virus production may vary depending on the type of cell used for the propagation of the virus. Standard techniques for detecting proteins, such as Western-Blot analysis, or the SRD assay for influenza virus, or CPE as described above, can also be used to determine the optimum range of cell densities required to obtain an optimized viral yield.
In order to produce large amounts of virus, it may also be advantageous to implement a virus adsorption phase. By "adsorption phase" is meant that the cells are inoculated with the virus at a high density, so as to promote the adsorption of the virus on them. cell membranes for a short period of time before the cell density is reduced for the rest of the infection period and until the virus is collected. For example, inoculation of cells with the virus is practiced when the cell density is at least 8 x 10 6 cells / ml, advantageously at least 9 x 10 6 cells / ml, more preferably at least 10 x 10 6 cells / ml, more preferably at least 11 x 10 6 cells / ml, more preferably at least 12 x 10 6 cells / ml, more preferably at least 13 x 10 6 cells / ml or more, such as x 10 6 cells / ml, 25 x 10 6 cells / ml, or 30 x 10 5 cells / ml. Advantageously 30 min, more preferably 45 min or 1 h, 1:30, or 2h after inoculation with the virus, the inoculated cells are diluted by a factor ranging from 2 to 5, advantageously 3, for the rest of the infection process, ie for subsequent incubation before the virus produced is collected. Alternatively, at the end of the adsorption phase, the inoculated cells are diluted so as to obtain a final cell density ranging from 3 to 5 × 10 6 cells / ml, advantageously 4 × 10 6 cells / ml, for the rest of the infection process, ie for subsequent incubation before the virus produced is collected.
In another embodiment, when the cell density is at least 8 x 10 6 cells / ml, at least 9 x 10 6 cells / ml, at least 10 x 10 6 cells / m, at least 11 X 106 cells / ml, of at least 12 x 106 cells / ml, of at least 13 x 106 cells / ml, of at least 20 x 106 cells / ml, of at least 25 x 106 cells / ml, or at least 30 x 106 cells / ml, the cells are inoculated with the virus, and immediately after inoculation, the inoculated cells are diluted, either by a factor of 2 to 5, suitably 3, or to obtain a final cell density ranging from 3 to 5 × 10 6 cells / ml, suitably 4 × 10 6 cells / ml, for the rest of the infection process, ie for a subsequent incubation before the virus produced is collection. .
When an adsorption phase is implemented, the input power density may suitably be kept low, such as, for example, from 2 to 10 W / m 3, from 4 to 8 W / m 3. m3, advantageously 7 W / m3, during said phase, ie after the cells have been inoculated with the virus of interest and before the inoculated cells are diluted. Therefore, in one embodiment of the invention, the method of producing a virus in cell culture comprises at least the steps of a) using a population of cultured cells in a culture medium, b) infecting the population of cells with the virus by i. inoculation of the population with the virus when the cell density is at least 8 x 10 cells / ml, at least 9 x 10 6 cells / ml, at least 10 x 10 6 cells / ml, at least 11 x 10 6 cells / ml, at least 12 x 10 6 cells / ml, or at least 13 x 10 6 cells / ml for 30 min, 45 min, 1 h, 1 h 30, or 2 h, then dilution of the cells inoculated by a factor ranging from 2 to 5, or a factor of 3, or alternatively, such that the final cell density obtained is from 3 to 5 x 10 6 cells / ml, suitably 4 x 10 6 cells / ml and ii. incubating the inoculated population diluted to allow the virus to replicate and propagate, the input power density applied to the cell culture being at least 15 W / m3, at least 30 W / m3, at least 60 W / m3, at least 100 W / m3, or at least 120 W / m3, before the cells are inoculated with the virus and reduced to a value of 2 to 10 W / m3, from 4 to 8 W / m3, or 7 W / m3, after the cells have been inoculated and before the inoculated cells are diluted, and increased to at least 15 W / m3, at least 30 W / m3 at least 60 W / m 3, at least 100 W / m 3, or at least 120 W / m 3 after the inoculated cells have been diluted and until the produced virus is collected. Alternatively, the input power density can be maintained at a constant value throughout the adsorption phase. Therefore, in another embodiment, the input power density applied to the cell culture according to the present invention is maintained at least 15 W / m 3, at least 30 W / m 3, at least 60 W / m 3, minus 100 W / m 3, or at least 120 W / m 3, during the successive steps of using a population of cells cultured in a cell culture medium, inoculating said population with the virus of interest for 30 min, 45 min, lh, lh30, or 2h, dilution of the inoculated cells according to the above conditions, and incubation of the diluted inoculated population. Inoculation can be performed at a MOI (Multiplicity of infection) of about ICh1 at 10 ~ 7, advantageously about 10 -2 to 10-6, and more preferably, about 10-5. .
The temperature and pH conditions for virus infection may vary. The temperature can range from 32 to 39 ° C depending on the type of virus. For the production of the influenza virus, infection of the cell culture may vary depending on the strain that is produced. The influenza virus infection is advantageously carried out at a temperature of from 32 to 35 ° C, more preferably at 33 ° C. In one embodiment, the virus infection occurs at 33 ° C. In another embodiment, the virus infection occurs at 35 ° C. Proteases, typically trypsin, can be added to the cell culture according to the strain of the virus to allow replication of the virus. The protease can be added at any appropriate stage during cultivation. Trypsin is preferably of non-animal origin, i.e. the protease is not purified from an animal source. It is conveniently recombinantly produced in a microorganism, such as a bacterium, a yeast or a plant. As suitable examples of recombinant trypsin, there is Trypzean, a recombinant trypsin produced in wheat (Prodigen, 101 Gateway Blvd, Suite 100 College Station, Texas 77845. Manufacturer Code: TRY), or TrpLE (Invitrogen) which is a trypsin-like enzyme expressed in a fungus (WO2004 / 020612). In one embodiment, trypsin is added at the same time as the virus is inoculated to cells, i.e. trypsin is added at day 0 (J0). In another embodiment, trypsin is further added at different time points after inoculation, such as, for example, on day 1 (J1) and / or day 4 (J4) after inoculation. In another embodiment, trypsin is further added daily after virus inoculation until the virus produced is collected. . '
Once infected, the cells can release newly formed viral particles into the culture medium by spontaneous lysis of the host cells, also called passive lysis. Therefore, in one embodiment, the viral harvest produced by the cells can be obtained at any time after virus inoculation, by harvesting the cell culture medium. This mode of harvest is particularly appropriate when it is desired to harvest the virus produced by the cells at different time points after virus inoculation, and group the different crops, if necessary.
Alternatively, after infection with the virus, the cell culture-based virus can be harvested using an external factor for lysis of host cells, also called active lysis. However, unlike the previous, this method of harvesting requires that the viral harvest derived from the cells is collected at a single time point, insofar as the active lysis immediately ends the cell culture.
Methods that can be used for active cell lysis are known to those skilled in the art. Useful methods in this regard are, for example, freeze-thawing, solid phase shearing, hypertonic and / or hypotonic lysis, liquid phase shearing, high pressure extrusion, detergent lysis, or any combination thereof. of these.
According to one embodiment, the cell culture-based viral harvest can be obtained at any time after virus inoculation by harvesting cell culture supernatants, lysing the inoculated cells, or both.
Before harvest, the infection of the cells can last from 2 to 10 days. According to a specific embodiment, the culture supernatants of days 3, 4 and 5 after inoculation are harvested and pooled for subsequent downstream treatment (isolation of the virus or purification of the virus). In another embodiment, the supernatant of the cell culture is collected from day 5 after inoculation. The optimal time to harvest the virus produced by the cells is usually based on the determination of the peak of infection. For example, CPE (cytopathic effect) is measured by monitoring morphological changes in host cells after virus inoculation, including rounding, disorientation, swelling or shrinkage, death, and detachment. of the cell surface. Detection of a specific viral antigen can also be monitored by standard protein detection techniques, such as Western Blot analysis. Harvesting can then be performed when the desired detection level is reached. In the particular case of the influenza virus, the HA content can be monitored at any time after the cells are inoculated with the virus by the SRD assay (Wood, JM, et al (1977) J. Biol Standard. 5, 237-247), which is a technique known to those skilled in the art. In addition, the SRD assay can also be used to determine the optimal cell density range required to achieve optimized viral yield.
In the context of the present invention, it will be understood that the cell culture phase comprises any step preceding the virus collection step, while the virus purification phase comprises any step following said collection step.
According to the invention, after production in cell culture, the virus is purified. Any suitable step or technique known in the field of virus purification can be conveniently implemented in the method according to the invention after the produced virus is collected. In one embodiment, the method according to the invention comprises at least one step selected from clarification, ultrafiltration / diafiltration, ultracentrifugation and chromatography of the viral harvest, or any combination thereof.
In a specific embodiment, during the virus purification phase, the method according to the invention comprises at least one step of clarifying the viral harvest, an ultrafiltration / diafiltration step to thereby obtain a retentate and a step of ultracentrifugation.
After collecting the cell culture medium containing the virus from the infected cells, the viral harvest obtained is typically clarified to separate the virus from the cellular material, such as intact cells or cell debris. The clarification can be carried out by a filtration step, typically a microfiltration step, i.e. using filters having a pore size typically between 0.1 and 10 μm. Suitable filters can use cellulose filters, regenerated cellulose filters, cellulosic fibers combined with inorganic filtration aids, a cellulose filter combined with inorganic filtration aids and organic resins, or any combination thereof , and polymer filters. Although not required, a multiple filtration process may be implemented, such as a two or three step process consisting of, for example, sequentially and progressively removing impurities based on their size, using filters having an appropriate nominal pore size, in particular, filters having a nominal pore size decreasing, allowing to begin by removing large precipitates and cell debris. In addition, one-step operations using a relatively tight filter and centrifugation can also be used for clarification. More generally, any clarification approach including, among others, direct flow filtration or "frontal" filtration, depth filtration, tangential flow filtration or. Cross-flow filtration, or centrifugation, which provides a filtrate of sufficient clarity not to foul the membrane and / or the resins in subsequent steps, will be acceptable for use in the clarification step of the present invention. In one embodiment, the viral clarification is carried out by deep filtration, in particular, using a three-stage filtration composed, for example, of three different depth filters having nominal porosities of 5 pm-0. , 5 pm - 0.2 pm. In another embodiment, the viral harvest is clarified by microfiltration, optionally preceded by a centrifugation step as a pre-clarification. In particular embodiments in which the process for producing a virus, such as influenza virus, according to the invention comprises a microfiltration-type clarification step during purification step d), the viral yield, such as the HA yield for the influenza virus, obtained after said clarifying step is at least 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75 %, 76%, 77%, 78%, 79%, 80%, 85%, 90% or more.
According to the present invention, during the virus purification phase of the method according to the present invention, the viral harvest can also be subjected to ultrafiltration (sometimes called "diafiltration" when it is used to replace the buffer), to concentrate the virus and / or replace the buffer. This step is particularly advantageous when the virus to be purified is diluted, as is the case, for example, when the pooled viral crops are collected by infusion over several days after inoculation. The method used to concentrate the virus and / or replace the buffer according to the method of the present invention may include any filtration method in which the concentration of the virus is increased by forcing the diluent to pass through a filter so that the diluent is driven from the virus suspension while the virus, unable to cross the filter, thus remains in concentrated form in the viral preparation. . If membranes or filters which are not neutral but positively charged are used, it may be useful to carry out an additional step of rinsing said membrane or said filter with a rinse buffer comprising salts to elute the viral fraction which may have been selected because of ionic interactions with the membrane or filter. An example of a suitable salt that can be included in the rinse buffer is sodium chloride (NaCl), which can be present in a concentration ranging from 0.1 to 2M, in particular from 0.5 to 1.5M , an advantageous way of IM. In an embodiment according to the invention, when the clarification is carried out by membrane filtration, whether it is a pre-clarification or a clarification, said clarification comprises a membrane rinsing step at the same time. using a buffer comprising NaCl, in particular, 1M NaCl. Ultrafiltration can include diafiltration, which is an ideal way of removing and replacing salts, sugars, non-aqueous solvents, removing low molecular weight materials, or rapidly replacing ionic and / or ionic environments. pH. The microsolutes are removed very effectively by adding solvent to the solution being ultrafiltered at a rate equal to the ultrafiltration rate. The micro-species are washed from the constant volume solution, isolating the retained virus. Diafiltration is particularly advantageous when a downstream step requires the use of a specific buffer to obtain. an optimal reaction. For example, the implementation of a diafiltration step prior to degradation of the host cell nucleic acids with an endonuclease may allow the endonuclease reaction to proceed in a specific buffer and optimal for the endonuclease. The concentration and the diafiltration can also be implemented at any appropriate stage of the purification process, when it is desired to remove undesirable compounds, such as sucrose, after a sucrose gradient ultracentrifugation, or such as formaldehyde, after a step inactivation of the virus with formaldehyde. The system consists of three distinct process streams: the loaded solution (containing the virus), the permeate and the retentate. Depending on the application, filters with different pore sizes may be used. In the present invention, the retentate contains the virus and can be used for further purification steps, if desired. The composition of the membrane may be, inter alia, regenerated cellulose, polyethersulfone, polysulfone, or derivatives thereof. The membranes can be flat sheets (also called "flat screens") or hollow fibers.
In one embodiment, the virus purification phase in the method according to the invention comprises at least one. ultrafiltration / diafiltration step, advantageously at least two ultrafiltration / diafiltration steps.
According to the application for which the virus produced in cell culture is purified ,. it may be desirable to also remove nucleic acid contaminants from the host cells from the virus harvest. In particular, when the purified virus is to be incorporated into a vaccine, the nucleic acids of the host cells must be degraded and removed from the host. purified virus. The degradation of nucleic acids is frequently carried out by using nucleases targeting RNA and DNA. A non-limiting example of a nuclease suitable for degrading nucleic acids of host cells is Benzonase ™. Benzonase ™, or any other appropriate nuclease, may be added at any stage appropriate to the virus purification process. In one embodiment, the method according to the invention comprises a nuclease degradation step, advantageously a Benzonase ™ treatment. For example, a nuclease may be added to the retentate obtained after ultrafiltration of a clarified cell culture medium containing the virus. Alternatively, the nucleic acid degradation of the host cells may be effected by a virus inactivation step with a beta-propiolactone.
If desired, the virus obtained according to the present invention may further be purified using standard techniques used for the purification of viruses such as density gradient centrifugation, for example sucrose gradient ultracentrifugation and / or chromatography, such as than ion exchange chromatography.
In one embodiment, the method according to the invention comprises at least one step of sucrose gradient centrifugation. In another embodiment, the purification phase of the process according to the invention comprises at least one clarification step, such as, for example, a centrifugation step followed by a microfiltration step, at least one step of ultrafiltration, and a sucrose gradient centrifugation step. In particular embodiments in which the process for producing a virus, such as influenza virus, according to the invention comprises at least one step of sucrose gradient centrifugation during step d) purification, the ability to load on the rotor of the centrifuge is at least 40, at least 50, at least 60, or more, liters of viral crop, or the "equivalent" amount, per liter of rotor, and the viral yield , such that the HA yield for the influenza virus obtained after said sucrose gradient centrifugation step is at least 65%, 66%, 67% ·, 68%, 69%, 70%, 71%, 72% , 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90% or more.
According to the process of the invention, it is possible to combine a purification step, such as a sucrose gradient ultracentrifugation, with a step. fragmentation of viruses. In particular, a fragmentation agent may be added to the sucrose gradient. This embodiment is particularly suitable when it is desired to minimize the total number of steps of the process according to the invention, since it allows, in a single operation, both to purify and to fragment the virus. As a result, in some embodiments, when at least one sucrose gradient ultracentrifugation is performed, the sucrose gradient further comprises a fragmentation agent.
Alternatively, the virus fragmentation step of the method according to the present invention, when implemented, is performed in batch mode. At the end of the virus purification phase, the viral preparation obtained according to the method of the present invention can be suitably subjected to sterile filtration, a practice common in processes using pharmaceutical materials, such as immunogens or vaccines, and known to those skilled in the art. This sterile filtration may, for example, be suitably carried out by filtration of the 0.22 μm filter preparation. Once the preparation is sterile, the virus and viral antigens are ready for clinical use, if desired.
Immunogenic compositions, particularly vaccines, can generally be formulated as a subion, e.g. as a fragmented virus, the lipid envelope of which has been dissolved or disrupted, or in the form of one or more purified viral proteins (vaccine subunits). Alternatively, the immunogenic compositions may comprise a whole virus, e.g., a whole live attenuated virus, or an inactivated whole virus.
Virus fragmentation methods, such as influenza viruses, are well known in the state of the art (WO02 / 28422). The fragmentation of the virus is carried out by breaking or fragmenting the whole virus whether infectious (wild type or attenuated) or non-infectious (inactivated) using a breaking concentration of a fragmentation agent. The fragmentation agents generally comprise agents capable of breaking and dissolving the lipid membranes. Typically, the fragmented influenza virus has been produced by a solvent / detergent treatment, such as tri-n-butyl phosphate, or diethyl ether in combination with Tween ™ (fragmentation known as fragmentation). Tween-ether) and this process is still used in some production units. Other fragmentation agents currently used include detergents or proteolytic enzymes or bile salts, for example, sodium deoxycholate. Detergents that can be used as fragmentation agents include cationic detergents, eg cetyl trimethyl ammonium bromide (CTAB), other ionic detergents, eg sodium lauryl sulfate (SLS), taurodeoxycholate, or detergents nonionics such as Tween or Triton X-100, or the combination of any two or more detergents.
In one embodiment, the fragmentation agent is deoxycholate. In another embodiment, the fragmentation agent is Triton X-100. In yet another embodiment, the method according to the invention uses a combination of Triton X-100 and sodium lauryl sulphate as fragmentation agents.
The fragmentation process can be implemented in batch, continuous or semi-continuous mode. In batch mode, the fragmented virus may require an additional purification step, such as a chromatography step. It is not necessary to implement a fragmentation step alone, since it is possible to practice fragmentation simultaneously with a purification step.
For example, a detergent may be added to the sucrose gradient for purifying proteins by ultracentrifugation, as described above. In one embodiment, the method according to the invention comprises a step of fragmentation implemented in batch mode with a detergent, in particular Triton X-100, in addition to at least one homogenization step.
For the safety of the vaccines, it may be necessary to reduce the infectivity of the virus suspension during the various steps of the purification process. The infectivity of a virus is determined by its ability to replicate in a cell line. Therefore, the method according to the present invention comprises, optionally, at least one inactivation step of the virus. Inactivation can be carried out using, for example, beta-propiolactone (BPL), formaldehyde, or UV, or any combination thereof, at any appropriate stage of the process. In a specific embodiment, the method according to the invention further comprises at least one GLP treatment step. In a specific embodiment, the method according to the invention further comprises at least one GLP treatment step and at least one formaldehyde treatment step. Formaldehyde and GLP can be used sequentially, in any order, for example, formaldehyde is used after GLP. In one embodiment, the formaldehyde treatment is followed by at least one homogenization step. In particular, when UV is used as an inactivation method, the implementation of the homogenization of the viral preparation prior to UV exposure can contribute to improving the inactivation efficiency of the virus. Viruses, or parts of viruses, which might be inside aggregates, whether aggregates of viruses or clusters of viruses / cells, can escape radiation due to their burial within said aggregates, and therefore some viruses or parts of viruses are inaccessible to the inactivating agent. In one embodiment, the viral preparation obtained by the method according to the present invention is inactivated, for example, by UV radiation, and the homogenization is carried out immediately before said inactivation. The viral inactivation conditions may vary and will be determined, in particular, by evaluation of the residual viral infectivity by measuring the infectious dose in tissue culture (TCID / ml).
The immunogenic compositions according to the present invention, including the vaccines, may optionally contain the usual additives for the vaccines, in particular substances which increase the immune response elicited in the vaccines. a patient receiving the composition, i.e., said adjuvants.
In one embodiment, the immunogenic compositions that comprise a viral virus or antigen according to the present invention mixed with a suitable pharmaceutical vehicle are contemplated. In a specific embodiment, they comprise an adjuvant.
The adjuvant compositions may comprise an oil-in-water emulsion which comprises a metabolizable oil and an emulsifying agent. In order for any oil-in-water composition to be suitable for human administration, the oily phase of the emulsion system must include a metabolizable oil. The meaning of the term "metabolizable oil" is known in the state of the art. "Metabolizable" can be defined as "able to be metabolized" (Dorland's Illustrated Medical Dictionary, W. B. Sanders Company, 25th Edition (1974)). The oil can be any vegetable oil, fish oil, animal oil or synthetic oil, which is not toxic to the recipient and can be metabolized. Nuts, seeds, and grains are common sources of vegetable oils. Synthetic oils are also part of this invention and may include commercial oils such as NEOBEE® and others.
A particularly suitable metabolizable oil is squalene. Squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexene) is an unsaturated oil found in large quantities in shark, and in smaller amounts, in olive oil, wheat germ oil, rice bran oil, and yeast, and is a particularly preferred oil in the context of this invention. Squalene, which is an intermediate in the biosynthesis of cholesterol, is therefore a metabolizable oil (Merck-Index, 10th Edition, entry No. 8619). In another embodiment of the invention, the metabolizable oil is present in the immunogenic composition in an amount of 0.5. at 10% (v / v) of the total volume of the composition. . The oil-in-water emulsion further comprises an emulsifying agent. The suitable emulsifying agent may be polyoxyethylene sorbitan monooleate, In addition, said emulsifying agent is suitably present in the vaccine or immunogenic composition at 0.125 to 4% (v / v) of the total volume of the composition. The oil-in-water emulsion according to the present invention optionally comprises a tocol, tocols are known in the state of the art and described in EP0382271. A suitable tocol may be an alpha-tocopherol or a derivative thereof. ci such as alpha-tocopherol succinate (also known as Vitamin E succinate) .These tocol is suitably present in the adjuvant composition in an amount of 0.25 to 10% (v / v). of the total volume of the immunogenic composition.
The method of producing the oil-in-water emulsions is well known to those skilled in the art. Generally, it comprises mixing the oily phase (optionally containing a tocol) with a surfactant such as a PBS / TWEEN80 ™ solution, followed by its homogenization using a homogenizer, being understood for the human being. a process comprising passing the mixture twice through a syringe needle would be suitable for homogenizing small volumes of liquid. In the same way, the microfluidic emulsification process (M110S Microfluidics apparatus, maximum of 50 passes, for a period of 2 minutes at a maximum inlet pressure of 6 bar (outlet pressure of about 850 bar) ) could be adapted by those skilled in the art to produce more or less significant emulsion volumes. The adaptation could be carried out by conventional experimentation comprising measuring the obtained emulsion until a preparation having oil droplets of the required diameter is obtained.
In an oil-in-water emulsion, the oil and the emulsifier are in an aqueous vehicle. The aqueous vehicle may be, for example, a phosphate buffered saline solution.
In particular, the oil-in-water emulsion systems according to the present invention have a small submicron oil droplet size. Suitably, the droplet sizes will be in the range of 120 to 750 nm, more preferably 120 to 600 nm in diameter. More particularly, the oil-in-water emulsion contains oil droplets of which at least 70% in intensity have a diameter of less than 500 nm, more particularly at least 80% in intensity have a diameter of less than 300 nm, more particularly at least 90% in intensity has a diameter in the range of 120 to 200 nm.
The size of the oil droplets, i.e. their diameter, according to the present invention is indicated in intensity. There are several ways to determine the diameter of oil droplets by intensity. Intensity is measured using a calibration instrument, appropriately by dynamic light scattering such as Malvern Zetasizer 4000 or suitably Malvern Zetasizer 3000HS. A detailed procedure is indicated in Example II. 2. A first possibility is to determine the mean diameter Z ZAD by dynamic light scattering (PCS spectroscopy by photon correlation); this method can additionally give the polydispersity index (PDI), and the ZAD and the PDI are both calculated with the cumulants algorithm. These values do not require to know the refractive index of the particles. A second way is to calculate the diameter of the oil droplet by determining the size distribution of whole particles by another algorithm, the Contin, or NNLS, or the automatic "Malvern" (default algorithm supplied with the calibration instrument). Most of the time, as the refractive index of particles of a complex composition is unknown, only the distribution of intensities is taken into account, and if necessary the average of the intensities coming from this distribution.
The adjuvant compositions may further comprise a Toll-like receptor agonist (TLR) 4. By "TLR4 agonist" is meant a component that is capable of eliciting a signaling response through the TLR4 signaling pathway, either as a direct ligand or indirectly by. generation of an endogenous or exogenous ligand (Sabroe et al, JI 2003 pl630-5). TLR 4 may be a derivative of lipid A, more particularly monophosphorylated lipid A or more particularly 3-deacylated monophosphoryl lipid A (3D-MPL).
3D-MPL is marketed under the brand name MPL® by GlaxoSmithKline Biologicals North America and primarily promotes responses of CD4 + T cells with an IFN-γ (Th1) phenotype. It can be produced according to the methods described in GB 2 220 211 A. Chemically it is a mixture of monophosphorylated lipid A 3-deacylated with 3, 4, 5 or 6 acylated chains.
In particular, in the adjuvant compositions of the present invention, small particle 3D-MPL is used. The small particle 3D-MPL has a particle size such that it can be sterile filtered through a 0.22 μπι filter. These preparations are described in International Patent Application No. WO94 / 21292. Synthetic derivatives of lipid A are known and considered to be TLR4 agonists, these derivatives comprising inter alia: OM174 (2-deoxy-6-O- [2-deoxy-2 - [(R) -3-dodecanoyloxytetrazol) decanoylamino] -4-O-phosphono-β-D-glucopyranosyl] -2 - [(R) -3-hydroxytetradecanoylamino] -α-D-glucopyranosyl dihydrogenphosphate), (WO 95/14026) OM 294 DP (3S, 9R) - 3 - [(R) -Dodecanoyloxytetradecanoyl] amino] -4-oxo-5-aza-9 (R) - [(R) -3-hydroxytetradecanoylamino] decan-1,10-diol, 1,10-bis ( dihydrogenophosphate) (WO99 / 64301 and WO 00/0462) OM 197 MP-Ac DP (3S, 9R) -3 - [(R) -dodecanoyloxy-tetradecanoylamino] -4-oxo-5-aza-9 - [(R)) 3-hydroxytetradecanoylamino] decan-1,10-diol, 1-dihydrogenphosphate 10- (6-aminohexanoate) (WO 01/46127) Other TLR4 ligands which can be used are the alkylglucosaminide phosphates (AGP) such as those described in WO 98/50399 or US Pat. No. 6,303,347 (processes for the preparation of AGPs are also described), or pharm salts. aceutically acceptable AGP as described in US Patent No. 6,764,840. Some AGPs are TLR4 agonists, and others are TLR4 antagonists. Both should be useful as adjuvants. In addition, other suitable TLR-4 agonists are described in US Patents Nos. 2003/0153532 and 2205/0164988. The invention is particularly suitable for the preparation of immunogenic compositions based on influenza virus, comprising vaccines. Various forms of influenza virus are currently available. They are usually based on either a live virus or an inactivated virus.
Inactivated viruses can be based on whole virions, fragmented virions or purified surface antigens (including HA). Influenza antigens can also be in the form of virosomes (viral-type liposomal particles lacking nucleic acids).
The virus inactivation methods and fragmentation methods described above are applicable to the influenza virus.
Influenza virus strains that can be used in vaccines change from one season to another. In the current pandemic period, vaccines typically include two influenza A strains and one influenza B strain. Trivalent vaccines are typical, but a higher valence, such as quadrivalence, is also contemplated in the present invention. The invention may also utilize HA from pandemic strains (ie strains to which the vaccine recipient and the human population in general are immunologically naive), and influenza vaccines for pandemic strains may be monovalent or based on a normal trivalent vaccine supplemented with a pandemic strain.
The compositions according to the invention may comprise one or more antigens originating from one or more influenza virus strains, comprising the influenza A virus and / or the influenza B virus. In particular, a trivalent vaccine comprising antigens from two strains A virus and a strain of influenza B virus is contemplated by the present invention. Alternatively, a quadrivalent vaccine comprising antigens from two strains of virus A and two strains of influenza virus B is also within the scope of the present invention. ,
The compositions according to the invention are not limited to monovalent compositions, i.e. comprising only one type of strain, i.e. only seasonal strains or only pandemic strains. The invention also encompasses multivalent compositions comprising a combination of seasonal and / or pandemic strains. In particular, a quadrivalent composition, which may be added, comprising three seasonal strains and one pandemic strain is within the scope of the present invention. Other compositions within the scope of the invention include a trivalent composition comprising two strains A and one strain B, such as strains H1N1, H3N2 and B, and a quadrivalent composition comprising two strains A and two strains B of different lineages, such as H1N1, H3N2, B / Victoria and B / Yamagata. HA is the major immunogen in current inactivated influenza vaccines, and vaccine doses are normalized with reference to HA levels, typically measured by SRD. Existing vaccines typically contain about 15 μg of HA per strain, although lower doses may be used, e.g. in children, or in pandemic situations, or when adjuvant is used. Fractional doses corresponding to half (i.e. 7.5 μg of HA per strain) or one quarter were used, as well as higher doses, in particular doses x3 or x9. Consequently, the immunogenic compositions according to the present invention may comprise between 0.1 and 150 μg of HA per influenza strain, in particular between 0.1 and 50 μg, eg 0.1-20 μg, 0.1-15 μg. 0.1-10 μg, 0.1-7.5 μg, 0.5-5 μg, etc. Specific dosages include about 15, about 10, about 7.5, about 5 μg per strain, about 3.8 μg per strain, and about 1.9 μg per strain.
Once the influenza virus has been purified for a particular strain, it can be combined with viruses from other strains to obtain a trivalent vaccine, for example, as described above. It is more appropriate to treat each strain separately and to mix the monovalent masses to obtain a final multivalent mixture, rather than mixing viruses and degrading the DNA and purifying it from a multivalent mixture. The invention will be further described with reference to the following nonlimiting examples.
Example I: Influenza virus production using high input power density - EB66® cells were seeded in a 200 L single-use bioreactor (Sartorius AG Cultibag STR equipped with inclined blades, or a Rushton turbine, where appropriate) at a density of about 0.4 x 10 6 cells / ml in a total volume of 65 L, and 36.5 ° discontinuously cultured in suspension C at a stirring speed of 105 rpm corresponding to the application of an input power density of at least 30 W / m3 (No. 039, No. 048, No. 052, No. 058, No. 044, No. 046, and No. 043), or 135 rpm corresponding to the application of an input power density of at least 120 W / m3 (No. 053 and No. 057 ), or 65 rpm corresponding to the application of an input power density of at least 7 W / m3 (No. 025, No. 026, No. 027 and No. 031). The input power density of at least 30 W / m3 was obtained using the single-use bioreactor equipped with an impeller with inclined blades, while the input power density of at least 120 W / m3 m3 was obtained using the single-use bioreactor equipped with a Rushton turbine. After 3 days of growth, the cell density reached at least 9 × 10 6 cells / ml. At this point in time, which is considered as Day 0 (D0), the cells were inoculated with a solution comprising influenza H5N1 virus at a multiplicity of infection (MOI) of 1 x 10-5 and with trypsin (30 mPu / ml TrpLE from Invitrogen), and the temperature was raised to 35 ° C. One hour after inoculation, the inoculated cell suspension was diluted by a factor of 3 by adding fresh medium to a total volume of 200 L. During the 1:30 hours above, the input power density was has been reduced to 7 W / m3 (No. 039, No. 048, No. 052, No. 058, No. 044, No. 046,
No. 043, No. 053 and No. 057) is maintained at 7 W / m3 (No. 025, No. 026, No. 027 and No. 031). - Immediately after the inoculated cells have been diluted, the input power density has been increased to at least 30 W / m3 (No. 039, No. 048, No. 052, No. 058,
No. 044, No. 046, and No. 043), or at least 120 W / m3 (No. 053 and No. 057) and maintained at these levels, or maintained at 7 W / m3 (No. 026, No. 027 and No. 031), until the culture medium containing the virus is collected, 5 days after virus inoculation, to thereby obtain the viral harvest. Trypsin (10 mPu / ml of TrpLE from
Invitrogen) was further added on Day 1 (Day 1) and Day 4 (Day 4) after inoculation of the virus. The viral harvest was then purified as described above.
Example II: Effect of High Input Power Density on Microfiltration - Once harvested, the viral harvest was pre-clarified by continuous centrifugation at 10,500 rpm at 90 L / hr, thereby obtaining the pre viral harvest. -clarifiée. - The pre-clarified crop was then subjected to a microfiltration step using a 0.45 μm flat sheet membrane (Sartorius AG), 3 to TMP (constant transmembrane pressure) and flow rate (3 psi). and 600 L / m2 / h, respectively), thereby obtaining the clarified viral harvest. The influenza virus yield was evaluated during the microfiltration step by measuring the HA content before and after said step according to the SRD assay, as described below in Example IV. The results are shown in Table 1 as percentages to be compared to the 100% control value representing the total amount of HA present in the starting material, i.e. present in the pre-clarified viral harvest prior to microfiltration.
Table 1 - Efficiency of HA after microfiltration - Low power versus high volumetric power | §ppej | ^: ^ Pufssaÿ Sj &amp; '^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ "" "", "" ", * -" * "%" ~ "ig £,; -" f,. Λ · -;)>. <Ç, .'C, ~ '· ^ "FvR, - -> ^;> v, -r, > -"> * &amp; J s03 S 30. 12 '67 71 wëèÊzz ±: MMÉ! Ï ____ 30 12,6 92 82 | p;: 05Ef 77 30 11 82 81
Average vA./ '80 80 E. type 7' - !; ## EQU1 ##. ## EQU2 ## , v _________________
Mean Γ Γ 84 67 E. -type ** '- <- 5 m-o2 $ m * "13 75 ~ 41
SiP ^ i ^ S 7 11 57 91
SlSoSsif 7 9 ^ 3 82 77 m ~ ° 9ÎB 7__13 81 44
Mean 74 33 E.-type ** f * -'ipc '· 25 * pi: after 11 virus inoculation **: E. type: Standard deviation Results - Conclusions.
Although the increase in input power density during the cell culture phase (30 W / m3 vs. 7 W / m3) had no impact on the yield of HA present in the collected virus crop (see Table 1, fourth column, "J5 pi viral production"), a positive impact was observed on the yield of the HA obtained after the subsequent microfiltration step. Not only is the efficiency of HA obtained after microfiltration higher when the input power density is higher (see the respective Mean lines), but also the values obtained from one experiment to another are more regular and less variable ( see the respective E.-type lines - 6 to 25 when the input power density is 30 or 7, respectively). These results indicate that higher input power density during the upstream cell culture phase yields improved and more consistent HA yields during the downstream virus purification phase.
Example III: Effect of high input power density on sucrose gradient ultracentrifugation - The cells were cultured and infected with influenza H5N1 virus, and the virus was harvested as described in Example I above. above. The viral harvest was pre-clarified, and the pre-clarified crop was microfiltered as described in Example II. . The clarified viral harvest was then concentrated 10 times by ultrafiltration using a 750 Kd polysulfone hollow fiber membrane (GE Healthcare) and diafiltered against 5 volumes of PBS containing 125 mM of citrate pH 7. 4 to TMP (transmembrane pressure) and constant flow (3 psi and 35 L / m2 / h, respectively). - The ultrafiltration retentate was then subjected to. a sucrose gradient ultracentrifugation step, wherein the virus and contaminants migrate in the gradient to their respective density. The influenza virus has a density of about 1.19 g / cm3 and will therefore sediment in the gradient where the sucrose concentration equals the density of the virus (about 43% sucrose). a continuous linear gradient of 0 to 55% sucrose in the centrifuge rotor, the ultrafiltration retentate was loaded into said rotor at a retentate equivalent feed capacity corresponding to 30 L of harvest per rotor L (No. 039, No. 043, No. 032, No. 033,
No. 028 and No. 029), or 50-60 L of crop per rotor L (No. 044A, No. 044B, No. 046, No. 048, No. 025, No. 026, No. 027,
No. 031), as shown in Tables 2 and 3. When the entire sample is loaded, a 1-h banding time allowed the virus to reach its density and concentrate in the sucrose gradient before the centrifuge is stopped and the sucrose gradient is unloaded and fractionated. Viral particles were concentrated in a few fractions. The product fractions were in pH 7.4 PBS containing 125 mM citrate and sucrose. The purified whole virion was pooled from the sucrose percentage ranging from about 30 to 50%. This range has been determined based on the profiles. obtained by SDS-PAGE and by Western Blot analysis using anti-HA antibodies. The influenza virus yield was evaluated during the sucrose gradient ultracentrifugation step by measuring the HA content before and after said step according to the SRD assay, as described below in Example TV. The results are shown in Table 2 and Table 3 as percentages to be compared to the 100% control value representing the total amount of HA present in the starting material, ie present in the retentate. ultrafiltration before ultracentrifugation on sucrose gradient.
Table 2 - Yield of HA after Sucrose Gradient Ultracentrifugation at a Load Capacity of 30 L of Harvest per L of Rotor
*: E. type: Standard deviation
The indicated loading capacity (30) corresponds to the equivalent of ultrafiltration retentate in liters of viral harvest collected during step c) of the process according to the invention charged per liter of rotor of the centrifuge. Results - Conclusions At a load capacity of 30 L of harvest per L of rotor, the average yield of HA obtained after sucrose gradient ultracentrifugation is in the same acceptable range, ie 72-82%, as the power density of the reactor. input is 7 or 30 V / m3.
Table 3 - Yield of HA after sucrose gradient ultracentrifugation at a loading capacity of 50-60 L of crop per L of rotor
*: E. type: Standard deviation
The indicated loading capacity (50 or 60) corresponds to the equivalent of ultrafiltration retentate in liters of viral crop collected during step c) of the process according to the invention charged per liter of rotor of the centrifuge. Results - Conclusions At a higher load capacity of 50-60 L of crop per rotor L, when the input power density is 7 V / m3, ie at a low input power density, the HA yield The average obtained after sucrose gradient ultracentrifugation dropped significantly (see Table 3, 31%), as opposed to the yield of HA obtained at a load capacity of 30 L of harvest per L of rotor at one time. low power density of similar input (see
Table 2, 82%). On the other hand, when the input power density has been increased to 30 V / m3, the average HA yield obtained after sucrose gradient ultracentrifugation. the higher load capacity of 50-60 <L of crop per rotor L was kept within the same acceptable range (see Table 3, 71%). This indicates that the increase in input power density during the cell culture phase (30 W / m3 vs. 7 W / m3) allows twice as much harvest volume, or crop volume equivalent, to be loaded. , on the rotor used for the sucrose gradient ultracentrifugation step while maintaining the same acceptable range of HA yield. The load capacity on a centrifuge directly impacts the number of centrifuges required to operate on a large scale. The higher the load capacity, the lower the number of centrifuges required.
Example IV: SRD method used to measure the HA content -
Glass plates (12.4-10 cm) were coated with an agarose gel containing a serum antiinfluenza serum concentration that is recommended by NIBSC. After taking the gel, 72 sample wells (3 mm in diameter) were formed in the agarose. 10 μl of appropriate dilutions of the reference and the sample were loaded into the wells. Plates were incubated for 24 hours at room temperature (20-25 ° C) in a humid chamber. The plates were then immersed overnight in NaCl solution and washed briefly in deionized water. The gel was then pressed and dried. When completely dry, the plates were stained with Coomassie brilliant blue solution for 10 minutes and decolorized twice in a mixture of methanol and acetic acid until well-defined colored areas appeared. After drying the plates, the diameter of the colored areas was measured in two directions at right angles. Alternatively, equipment for measuring the surface may be used. The dose-response curves for antigen dilutions as a function of surface area were plotted and the results were calculated using standard slope ratio assay methods (Finney, DJ (1952).) Statistical Methods in Biological Assay. Griffin, cited in Wood, JM, et al (1977) J. Biol Standard, 5, 237-247).

权利要求:
Claims (27)
[1]
CLAIMS AMENDED
A method of producing a virus in cell culture comprising at least the steps of: a) using a population of cells in a cell culture medium; b) infecting the cell population by: i. inoculation of the population with the virus, and ii. incubation of the inoculated population to allow the virus to replicate and spread, c) collect the virus produced, thereby obtaining a viral harvest, and d) purify the virus, in which an input power density of at least 15 W / m3, at least 30 W / m3, at least 60 W / m3, at least 100 W / m3, or at least 120 W / m3 is applied to the cell culture at least least during step b).
[2]
The method of claim 1, wherein the input power density is applied to the cell culture during step a) and step b).
[3]
The method of claim 1 or 2, wherein the cell density of the cells is at least 8 x 10 6 cells / ml, at least 9 x 10 6 cells / ml, at least 10 x 10 6 cells / ml, at least 11 x 106 cells / ml, at least 12 x 10 6 cells / ml or at least 13 x 10 6 cells / ml before the cell population was inoculated with the virus.
[4]
4. The method of claim 3, wherein during step b) the inoculated cells of step i. are diluted by a factor of 2 to 5, immediately after the virus has been inoculated, and left as is po &amp; 4 / C subsequent incubation.
[5]
5. The method according to claim 3, wherein during step b) the inoculated cells of step i. are diluted to obtain a cell density of from 3 to 5 × 10 6 cells / ml immediately after the virus has been inoculated, and left as is for subsequent incubation.
[6]
6. The method according to claim 3, wherein during step b) the virus is incubated for 30 min, 45 min, 1 h, 1 h 30, or 2 h after inoculation, before the inoculated cells are diluted by a factor ranging from from 2 to 5, and left as is for subsequent incubation.
[7]
The method according to claim 3, wherein during step b) the virus is incubated for 30 min, 45 min, 1 h, 1 h 30, or 2 h after the inoculation, before the inoculated cells are diluted to obtain a cell density ranging from 3 to 5 × 10 6 cells / ml, and left as is for subsequent incubation.
[8]
The method of claim 6 or 7, wherein during step b) the input power density is reduced to 2-10 W / m3, 4-8 W / m3, or 7 W / m3 and maintained at that level after inoculation of the cells and until the inoculated cells are diluted.
[9]
The method of claims 1-8, wherein trypsin is added to the cells during step b).
[10]
The method of claim 9, wherein the trypsin is added at the same time as the inoculation of the virus.
[11]
The method of claim 10, wherein trypsin is further added on day 1 and / or day 4 after inoculation.
[12]
The method of claim 10, wherein the trypsin is further added daily after virus inoculation until the virus produced from step c) is collected. ,
[13]
The method of claims 1 to 12, wherein the virus produced from step c) is collected between 2 to 10 days after virus inoculation.
[14]
The method according to claims 1 to 13, wherein the step of purifying the virus d) comprises at least one step selected from clarification, ultrafiltration / diafiltration, ultracentrifugation and chromatography of the viral harvest, or any combination of these.
[15]
The method of claim 14, wherein the step of purifying the virus d) comprises at least one step of clarifying the viral harvest.
[16]
The method of claim 15, wherein the viral harvest is clarified by microfiltration.
[17]
The method according to claims 14 to 16, wherein the step of purifying the virus d) comprises at least one step of sucrose gradient ultracentrifugation.
[18]
The method of claims 1 to 17, wherein the step of purifying virus d) comprises a step of inactivating the virus. έλ
[19]
19. The method of claim 18, wherein the inactivation step of the virus is carried out with beta-propiolactone.
[20]
The method of claims 1 to 19, wherein the step of purifying virus d) comprises a fragmentation step.
[21]
21. The method of claims 1 to 20, further comprising a step of formulating the purified virus in a vaccine.
[22]
22. The method of claims 1 to 21, wherein the virus is influenza virus.
[23]
23. The method of claim 22, wherein the influenza virus is subtype H2, H5, H6, H7 or H9.
[24]
24. The method of claim 22, wherein the influenza virus is subtype H1, H3 or B.
[25]
25. The method of claims 1 to 24, wherein the cells are mammalian or avian cells.
[26]
The method of claims 1 to 25, wherein the cells are cultured in suspension.
[27]
27. The method of claims 1 to 26, wherein the cells are EB66® cells.

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

WO2002028422A2|2000-10-02|2002-04-11|Glaxosmithkline Biologicals S.A.|Split enveloped virus preparation for intranasal delivery|

---

EP1985305A1|2007-04-24|2008-10-29|Vivalis|Duck embryonic derived stem cell lines for the production of viral vaccines|

---

WO2010052214A2|2008-11-05|2010-05-14|Glaxosmithkline Biologicals S.A.|Novel method|

---

WO2011012725A1|2009-07-31|2011-02-03|Baxter Healthcare S.A.|Method of producing a polypeptide or virus of interest in a continuous cell culture|

---

US4912094B1|1988-06-29|1994-02-15|Ribi Immunochem Research Inc.|Modified lipopolysaccharides and process of preparation|

---

US5192539A|1988-07-21|1993-03-09|Akzo N.V.|Infectious bursal disease virus production in continuous cell lines|

---

EP0382271B1|1989-02-04|1994-12-21|Akzo Nobel N.V.|Tocols as adjuvant in vaccine|

---

EP0812593B8|1993-03-23|2010-11-10|SmithKline Beecham Biologicals S.A.|Vaccine compositions containing 3-0 deacylated monophosphoryl lipid a|

---

CA2175375C|1993-11-17|2010-01-05|John Gwynfor Davies|Glucosamine disaccharides, method for their preparation, pharmaceutical composition comprising same, and their use|

---

DE19612966B4|1996-04-01|2009-12-10|Novartis Vaccines And Diagnostics Gmbh & Co. Kg|MDCK cells and methods of propagating influenza viruses|

---

US6764840B2|1997-05-08|2004-07-20|Corixa Corporation|Aminoalkyl glucosaminide phosphate compounds and their use as adjuvants and immunoeffectors|

---

US6303347B1|1997-05-08|2001-10-16|Corixa Corporation|Aminoalkyl glucosaminide phosphate compounds and their use as adjuvants and immunoeffectors|

---

US6113918A|1997-05-08|2000-09-05|Ribi Immunochem Research, Inc.|Aminoalkyl glucosamine phosphate compounds and their use as adjuvants and immunoeffectors|

---

EP0924298A1|1997-12-18|1999-06-23|Stichting Instituut voor Dierhouderij en Diergezondheid |Protein expression in baculovirus vector expression systems|

---

HU0102332A3|1998-06-08|2002-11-28|Sca Emballage France|Fast flattening packaging|

---

CZ302062B6|1998-06-30|2010-09-22|Om Pharma|Acyl dipeptide derivative process for its preparation, intermediates for the preparation thereof and pharmaceutical composition in which the derivative is comprised|

---

US20040006242A1|1999-02-01|2004-01-08|Hawkins Lynn D.|Immunomodulatory compounds and method of use thereof|

---

US6835721B2|1999-02-01|2004-12-28|Eisai Co., Ltd.|Immunomodulatory compounds and methods of use thereof|

---

WO2001046127A1|1999-12-22|2001-06-28|Om Pharma|Acyl pseudopeptides bearing a functionalised auxiliary spacer|

---

FR2808803B1|2000-05-11|2004-12-10|Agronomique Inst Nat Rech|MODIFIED ES CELLS AND SPECIFIC GENE OF ES CELLS|

---

DE60328802D1|2002-08-30|2009-09-24|Novozymes Inc|PROCESS FOR THE PREPARATION OF MAMMALS TRYPSINS|

---

EP1528101A1|2003-11-03|2005-05-04|ProBioGen AG|Immortalized avian cell lines for virus production|

---

FR2884255B1|2005-04-11|2010-11-05|Vivalis|USE OF EBX AVIATION STEM CELL LINES FOR THE PRODUCTION OF INFLUENZA VACCINE|

---

EP2350268B1|2008-11-03|2014-12-24|Crucell Holland B.V.|Method for the production of adenoviral vectors|

---

JP2012525830A|2009-05-08|2012-10-25|グラクソスミスクラインバイオロジカルズソシエテアノニム|Method for producing virus from cell culture including homogenization|KR101997329B1|2017-06-02|2019-10-02|전남대학교산학협력단|Method for Promoting Norovirus Replication|

---

US20200354661A1|2017-10-31|2020-11-12|Global Life Sciences Solutions Usa Llc|A flexible bag|

---

CN108261543B|2018-02-08|2019-03-08|华农（肇庆）生物产业技术研究院有限公司|The preparation method and applications of subculture cell source ND, IB, AI triple inactivated vaccine|

---

CN108187039A|2018-03-29|2018-06-22|山东信得动物疫苗有限公司|A kind of inactivated avian influenza vaccine production technology and product|

法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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US201361872024P| true| 2013-08-30|2013-08-30|

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US61/872024|2013-08-30|

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